PROJECT SUMMARY CD40 is a surface signaling molecule in the tumor necrosis factor receptor family that is typically expressed on B cells and other antigen-presenting immune cells, but has also been found on tumor cells and thus is implicated in the development of various cancers. One pathway central to CD40 signaling is the nuclear factor kappa B (NF-B) pathway, which is frequently dysregulated in cancers and has been shown to activate genes associated with cell proliferation, inflammation, angiogenesis, tumor promotion, metastasis, and suppression of apoptosis. The goal of this project is to provide a novel approach to study regulation of CD40-induced NF-B activation through post-translational modification of NEMO, the NF-B essential modulator, which may ultimately be used to identify new drug targets and/or strategies to improve upon current methods of cancer therapies. We have recently found that the mutation of two putative NEMO modification sites, 270 and 302, termed NEMO-DK, attenuate NF-B signaling to affect CD40-mediated B-cell functions in a mouse model. The following specific aims will address the central hypothesis that CD40-induced NEMO post-translational modification(s) play a critical physiological role in mediating NF-B activation in the course of CD40 signal transduction: 1) Elucidate the role of NEMO post-translational modifications in CD40-induced activation of NF- B; 2) Assess the biological impact of the NEMO-DK mutation in the context of CD40 signaling. The first aim will be accomplished using in vitro cell-based studies in primary mouse splenic B cells and cell line model systems to identify the CD40-induced NEMO modifications involving lysine residues 270 and/or 302 and assess the impact that mutation of either or both sites have on CD40-induced NF-B activation. The second goal will be achieved using a NEMO-DK knock-in mouse model to study in vivo B cell function, specifically NF- B target gene expression, CD40-induced cell proliferation and survival, and antigen-specific immunoglobulin class switch recombination, germinal center formation, and memory B cell formation. Fulfillment of these aims will extend our knowledge of CD40 signaling mechanisms that activate NF-B, contribute new tools and resources to propagate future research in this field with broader applications potentially in future drug screening of new compounds, and provide necessary insights into cancers and other malignancies exhibiting aberrant CD40 signaling that will facilitate development of better treatments and therapies.